1 Prezentare BIALOWIEZA septembrie 2008-final · 1 IFIN-HH National Insitutefor Physics and Nuclear...

1

IFIN-HH National Insitute for Physics and Nuclear Engeneering

Multipurpose Irradiation Facility IRASM

A Study on optimization the sample preparation for

gas chromatographic methods used in routine

irradiated foodstuffs detection

M. Virgolici, A.V. Medvedovici, M.M. Manea, M. Cutrubinis,

I.V. Moise, R.M. Georgescu

International Coference on

Recent Developments and Applications of Nuclear Technologies

Bialowieza, Poland

- 15 – 17 September 2008 -

METHODSIFIN-HH

Centrul de Iradieri Tehnologice IRASM

1. Using the Gas Chromatographic Microbial Identification System

(MIDI, Inc.) for fatty acids profiling of extracted fat from irradiated

foods. Enabling Cn-1:0 and Cn-2:1 hydrocarbons (HC) and 2-

alkylcyclobutanones prediction virtually from any fats which contain

fatty acids with chain length ranging from 9 – 20 carbon atoms in

length

2. Structure confirmation of main identified fatty acids by Gas

Chromatography coupled with Mass Spectrometry (GC-MS)

3. Cn-1:0 and Cn-2:1 HC detection using EN 1784 standard and

optimized GC-MS method for identification of irradiated foods that

contain fat

4. 2-Alkylcyclobutanones detection using EN 1785 standard and

optimized GC-MS method for identification of irradiated foods that

contain fat

5. Intercomparison with the ESR method for detection of irradiated

foods which contain bones

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Samples : Chicken, Salmon, Crab and Shrimp aquired from Romanian market

Samples storage: in freezer before and after irradiation

Gamma Irradiation : SVST Co-60 IRASM facility

Irradiation dosses : 0, 2, 7 and 10 kGy

Dosimetry : ECB (STDEV + 2% @ 1σ)

Fatty acids profiles and identification : Sherlock 6.0 Microbial Identification

System (MIDI, Inc. USA) and GC-MS performed on Agilent GC 6890N gas

chromatograph with two channels:

- classical split/splitless injector (S/SL) – Ultra 2 non polar capillary column –

classical Flame ionisation detector (FID) for Sherlock

- programable temperature vaporizer (PTV) CIS 4 (cooled injection systems)

made by Gerstel with Peltier cooling to 10C – HP-5ms non polar capillary

column – coupled with Agilent 5795 inert EI MSD (Mass Spectrometric Detector)

for structural identification using reference substances spectra and NIST 2005

MS Library

Samples and Instrumentation

2

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HC profiles and identification : performed on Agilent GC 6890N on both

channels (Flame ionisation and MS detection)

2-ACB identification : performed on Agilent GC 6890N on PTV GC-MS

channel

ESR free radical detection: Magnettech MiniScope 200 spectrometerMagnettech MiniScope 200 spectrometer

Samples and Instrumentation

Fatty acids profiling – Sherlock MIS 6.0IFIN-HH


Methylation

Saponification

NaOH solution water : methanol

HCl

methanolic solution

Pure Lipids

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Liquid – Liquid extraction

Cleanup

Extraction solvents

n-Hexane,

MTBE

NaOH

aqueous solution

Sherlock MIS GC-FID analysis

- injection hot split @ 250C

- i. d. 0,32mm, 25m long non-polar HP Ultra 2 capillary column

- 28K/min from 170C to 288C (~300 fatty acids in a window of 3.2 min)

- fast GC analysis (6min/sample)

- sample preparation time ~2h/15 samples

Fatty acids profiling – Sherlock MIS 6.0

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620:4 w6,9,12,15c

4661231

25

(18:2 w6,9c)18:2 w6,9c/18:0 ante (Linoleic acid)

172146323218:1 w9c (Oleic acid)

618418:1 w7c

13335618:0 (Stearic acid)

417:0

75516:1 w7c/16:1 w6c

30715202116:0 (Palmitic acid)

25114:0 (Myristic acid)

Shrimp

(MIDI)

Crab

(MIDI)

Salmon

(MIDI)

Chicken

(MIDI)

Chicken

(literature)

Fatty acid

FAME profiles

Fatty acids profiling – Sherlock MIS 6.0

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Fatty acids profiling – GC-MS

FA

ME

9:0

FA

ME

10:0

FA

ME

11:0

FA

ME

12:0

FA

ME

13:0

FA

ME

14:0

FA

ME

15:0

FA

ME

16:0

FA

ME

17:0

FA

ME

18:0

FA

ME

19:0

FA

ME

20:0

- GC reference substances : FAME C9:0 – C20:0 with concentrations of 0,01% and 0,005%

- injection: 1ul hot split @ 250C (PTV CIS 4) of same samples prepared by MIS procedures

- i.d. 0.25mm, 30m long non-polar HP-5ms capillary column; const. flow 1,3 ml/min

- 20K/min from 170C to 288C (fast GC analysis: 8min/sample)

Salmon meatIFIN-HH

2-tetradaca-5',8'-

dienylcyclobutanone1,7,10-C16:36,9-C17:212

18:2 w6,9c/18:0 ante

(Linoleic acid)

(2-TDCB)1,7-C16:28-C17:14618:1 w9c (Oleic acid)

C16:2C17:1818:1 w7c

(2-TCB)1-C16:1C17:0318:0 (Stearic acid)

C14:2C15:1516:1 w7c/16:1 w6c

(2-DCB)1-C14:1C15:01516:0 (Palmitic acid)

1-C12:1C13:0514:0 (Myristic acid)

2-ACBCn-2:1Cn-1:0

Composition

%Fatty acid

FA

ME

14:0 FA

ME

16:0

FA

ME

16:1

w7

c/1

6:1

w6

c

FA

ME

18:0

FA

ME

18:1

w9c

FA

ME

18:2

w6

,9c

4

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Fatty acids profiling – GC-MS

- MSD: transfer line @ 280C, EI source @ 230C, Quad @ 180C, PFTBA tune @ 1.3 ml/min

- for co-eluted peaks, MS spectral deconvolution was made with AMDIS v 2.62/2005

- spectral identification was made by means of reference substances (FAME), retention

time indexes based on Sherlock MIS callibration data, all correlated with MS spectra from

NIST05 Library

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Optimization of EN 1784 HC GC-MS detection method

GC-MS EN 1784 optimized classic chromatographic method:

- inj 1ul @ 250C – hot splitless (1min)

- temperature programme: 10K/min from 50C to 130C; 5K/min to 230C

- MSD detection : EI, SCAN 50-300amu

GC-MS EN 1784 PTV LVI chromatographic method:

- inj 5ul @ 10C – solvent vent @ 4ml/min (1min)

followed by splitless injection @ 300C (1.5min) & PTV cleanup @ 350C

- same temperature programme and MSD parameters

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10C was selected for minimizing the loss of analites during solvent evaporation in injector

1-C

12:1

C13:0

10C

20C

30C

40C

10C

20C

30C

40C

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- hot splitless : 1 ul @ 250C (1min)

- Calibration mixture 1ppm

C15:0

1-C

12:1

C13:0

1-C

14:1

1,7

-C16:2

1-C

16:1

C16:0

C17:0

C18:0

IST

DC

20:0

1-C

18:1

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- Solvent vent temperature optimization : @ 10C & 4ml/min

- Calibration mixture 1ppm

1-C

12:1

C13:0

1-C

14:1

C15:0

1,7

-C16:2

1-C

16:1

C16:0

C17:0

C18:0

IST

DC

20:0

1-C

18:1

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- solvent vent injection enables at least 10 times smaller concentrations

1UL

HOT SPLITLESS

5UL

SOLVENT VENT

0.1ppm

0.5ppm

6

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Optimization of EN 1784 HC GC-MS detection methodChicken Meat

2-tetradaca-5',8'-

dienylcyclobutanone1,7,10-C16:36,9-C17:231

18:2 w6,9c/18:0 ante

(acid linoleic)

(2-TDCB)1,7-C16:28-C17:13218:1 w9c (acid oleic)

(2-TCB)1-C16:1C17:0518:0 (acid stearic)

(2-DCB)1-C14:1C15:02016:0 (acid palmitic)

2-ACBCn-2:1Cn-1:0%Fatty acid

HC and 2-ACB

prediction

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1,7

-C16:2

1-C

14:1

C15:0

1,7

,10-C

16:3

1-C

16:1

6,9

-C17:2

8-C

17:1

C17:0

IS C

20:0

Chicken irradiated @ 10kGy

HOT SPLITLESS


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1-C

14:1

C15:0

1-C

16:1

6,9

-C17:2

8-C

17:1

C17:0

IS C

20:0

1,7

-C16:2

1,7

,10-C

16:3

Chicken irradiated @ 10kGy

Solvent vent method – same sample


7

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Salmon irradiated @ 10kGy

Solvent vent method

Optimization of EN 1784 HC GC-MS detection methodSalmon Meat

C15:0

1-C

14:1

1-C

12:1

6,9

-C17:2

8-C

17:1

1,7

-C16:2

1,7

,10-C

16:3

IS C

20:0

C17:0

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Optimization of EN 1785 2-ACB GC-MS detection method

GC-MS EN 1785 optimized classic chromatographic method:

- inj 1ul @ 250C – hot splitless (1min)

- temperature programme: 15K/min from 55C to 300C

- MSD detection : EI, SIM of m/z 98 (dwell 50ms) and m/z 112 (dwell 50ms)

GC-MS EN 1785 PTV LVI chromatographic method:

- inj 5ul @ 10C – solvent vent @ 4ml/min (1min)

followed by splitless injection @ 300C (1.5min) & PTV cleanup @ 350C

- same temperature programme and MSD parameters

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- hot splitless : 1 ul @ 250C (1min)

- 2-dodecylcyclobutanone (2-DCB) 0.25ppm

2-D

CB

IS 2

-CC

H

O

2-cyclohexylcyclobutanone

O

2-dodecylcyclobutanone

8

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- Solvent vent temperature optimization : @ 10C & 4ml/min

- 2-dodecylcyclobutanone (2-DCB) 0.25ppm (~ six times more sensitive than hot splitless)

2-D

CB

IS 2

-CC

H

O

2-cyclohexylcyclobutanone

O

2-dodecylcyclobutanone


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- solvent vent injection enables at least 10 times smaller concentrations


1UL

HOT SPLITLESS

5UL

SOLVENT VENT

0.025ppm

0.25ppm

better

linearity and sensitivity

Optimization of EN 1785 2-ACB GC-MS detection methodSalmon Meat

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2-D

CB

IS 2

-CC

H

2-T

CB

2-ACB inj hot splitless

MSD: SIM m/z 98 si m/z 112

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Optimization of EN 1785 2-ACB GC-MS detection methodSalmon Meat

2-ACB inj solvent vent – better sensitivity

MSD: SIM m/z 98 si m/z 112

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2-D

CB

IS 2

-CC

H

2-T

CB

2-DCB STRUCTURAL CONFIRMATIONSALMON MEAT

2-DCB NIST05

MSD: SCAN m/z 35 - m/z 300

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Component at scan 1931 (13.795 min) [Model = TIC] in C:\ msdchem\ 1\ DATA\ _LDAI_\ _SELECTIE-CONFERINTE-SEPT-2008\ QC-EN-1785-SOMON-10KGY-SCAN-SVENT.D\ data.ms2-Dodecylcyclobutanone

30 60 90 120 150 180 210 240

0

50

100

50

100

41

41

55

55

69

69

84

84

98

98

112

112

125

125

138

138

153166

166

192

194

207

210

220 238

238

2-TCB

MSD: SCAN m/z 35 - m/z 300

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(ma inlib ) Cyclobutanone, 2-tetradecyl-

50 70 90 110 130 150 170 190 210 230 250 2700

50

100

55

69

84

98

112

124138 152 166 180 194 222 238 248 266

O

(Text File) Component a t scan 2356 (15.161 min) [Model = +98u] in C:\ msdchem\ 1\ DATA\ _LDAI_\ _SELECTIE-CONFERINTE-SEPT-2008\ QC-EN-1785-SOMON-10KGY-SCAN-SVENT.D\ data.ms

30 60 90 120 150 180 210 240 2700

50

100

4155

69 83

98

112

125 140 157 173 193 211 238 266

2-TCB STRUCTURAL CONFIRMATIONSALMON MEAT

10

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Solvent extraction procedures

Standard extraction procedures involve time and high volumes of solvent:

- Soxhlet extraction : 150 ml/sample – 6hours extraction

Straight forward extraction techniques:

- Ultrasound fat extraction allows smaller extraction time, simple glassware, with the same volume of high purity solvents or smaller

- Direct solvent extraction method (Tewfik 2008) enables 3 times smaller solvent volumes, total reduction to sample preparation to 90 min, and the same results for 2-ACB.

Use of last two techniques could lead to comparable results in more resonabletime per sample and resonable costs.

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Detectie RES os pui

0

10000

20000

30000

40000

50000

60000

329 330 331 332 333 334 335 336 337 338 339

Camp magnetic (mT)

Inte

ns

itate

(u

.a.)

0 kGy 2 kGy 7 kGy 10 kGy

ESR method for detection of irradiated bonesDetection of irradiated Chicken

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Detectie RES os somon

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

55000

329 330 331 332 333 334 335 336 337 338 339

Camp magnetic (mT)

Inte

ns

ita

te (

u.a

.)


ESR method for detection of irradiated bonesDetection of irradiated Salmon

11

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Detectie RES os crab

0

10000

20000

30000

40000

50000

60000

70000

80000

90000

329 330 331 332 333 334 335 336 337 338 339

Camp magnetic (mT)

Inte

sit

ate

(u

.a.)


ESR method for detection of irradiated bonesDetection of irradiated Crab

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Detectie RES os crevete

0

10000

20000

30000

40000

50000

60000

70000

80000

90000

100000

329 330 331 332 333 334 335 336 337 338 339

Camp magnetic (mT)

Inte

ns

ita

te (

u.a

.)


ESR method for detection of irradiated bonesDetection of irradiated Shrimp

ConclusionsIFIN-HH


- Fatty acid profiling using Sherlock MIS method is an efficient way of

determination fatty acid composition of various fats before HC and 2-

ACB detection enabling use of standardized methods EN 1784 and EN

1785 outside of the available validation data

- Use of PTV Cooled Injection Systems can enable detection of smaller

quantities of HC and 2-ACB comparing with classical methods

- The sensitivity improvement of HC and especially 2-ACB detection in

conjunction with more faster extraction methods will enable a larger

number of samples for routine testing, and maybe smaller costs

- ESR method for detection of irradiated foodstuffs on bone samples

can be used for intercomparison purposes with chemical methods for

detection of meat samples

12

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Thank You for You Attention

Aspecte teoreticeProdusi chimici rezultati in urma iradierii lipidelor

IFIN-HH


Posibili produsi de radioliza ai

trigliceridelor

2-ACB

Reactii de formare

.....

IFIN-HH


Formarea 2-ACB.

Cele mai comune 2-ACB

prezente in alimente.

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